Environmental oestrogens have adverse effects on fish development and reproduction. The availability of models to study the differential effects of endogenous and exogenous oestrogens and to test for endocrine disrupting chemicals (EDCs) would be advantageous. Vitellogenins (VTGs) are precursors of egg yolk proteins and phospho-lipo-glycoproteins, and belong to a large lipid transfer protein superfamily. VTGs are synthesised differentially in the liver of females under the oestrogenic regulation of the oestrogen receptor (ER) and are distributed to oocytes via the circulatory system. During the last decade, in vitro systems such as fish cell lines have become very important in aquatic ecotoxicology. Thus, a zebrafish liver cell line (ZFL, CRL-2643) was developed for the investigation of VTGs and ERs induction potential oestrogens (E2/EE2). Surprisingly the levels of transcribed ERa and VTGs are very low in ZFL cells and cannot be induced by oestrogens. The weak ERa mRNA was proposed to be the crucial reason of negative VTG induction in responses of oestrogens. Therefore, a recombinant ERa (human Esrl) plasmid 'was subsequently constructed and transfected into ZFL cells. Due to the low efficiency of transfection, Esr 1 stable transfected ZFL cells were only obtained after a long period of selection. However, an unexplained protein band was detected in Esrl transfected cells by western blot. Conversely, the expression of Esrl protein was detected in Esrl transient transfected EcoPack cells (HEK-293) with higher transfection efficiency in parallel assays. Polycyclic aromatic hydrocarbons (PAHs) are suspected to have inhibitory effects on the expression of VTG and ER genes in fish liver through aryl hydrocarbon receptor (AhR) which is a ligand activated transcription factor stimulating the expression of the genes encoding xenobiotic metabolizing enzymes such as cytochrome P450-1A (CYP-IA). ~- naphthoflavone (bNF) is a potent agonist of AhR and also an inducer of such detoxification enzymes as CYPs. As expected, the prominent induction of CYP-IA was detected in ZFL cells by bNF exposure. This result indicated the characterization of hepatic mechanism in ZFL cells and initiated the investigation of the mechanism behind the cross-talk between ERs and AhRs under the combination exposure (bNF and EE2). The results of quantitative real-time, reverse-transcriptase polymerase chain reaction (qPCR) assays revealed that transcript levels of CYP-IA and AhR2 were increased by bNF exposure in a time- and concentration-dependent manner, but no significant effect in AhRls or ERs, whereas ER~2 seemed to be repressed by bNF. There are multiple VTG genes and an in silico analysis of their distribution in the zebrafish genome has identified eight genes (VTG-l to -8) and a pseudogene (VTG-9). A cluster of eight VTGs is located in a single 271 kbp region of chromosome 22, whereas a single gene (VTG-3) is mapped on chromosome 11. Sequence analyses of nucleotide and protein show extensive identity between VTG-l, -4 to -7 and -9 which share a domain structure (LvH- Pv-LvL), and VTG-2 is similar to -8 in terms of length and the presence of a von Willebrand Factor domain (LvH-Pv-LvL-vWFD) whereas VTG-3 is dissimilar in sequence and structural features since it lacks the phosvitin domain (LvH-LvL). qPCR assays were developed to measure transcripts of all VTG and ER genes in adult livers. Transcript levels of VTGs and ERs in vitellogenic female were ordered: VTG-l»VTG-2::::VTG-5>VTG- 4>VTG-6>VTG-7>VTG-3»VTG-8>VTG-9 and ERa>ER~2»ER~I, respectively. In male liver, transcript levels ofVTGs, ERa and ER~1 were undetectable or very low while ER~2 transcript was present in appreciable quantity. According to the genomic organization and phylogenetic analysis, it was speculated that the origin and evolution of multiple VTG genes in zebrafish was based on a number of whole genome duplications (WGDs), gene duplications and conversion events. Furthermore, tests for selection suggested that VTGs in zebrafish had undergone purifying selection in combination with mixed process of concerted and birth-and-death evolution.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:555819 |
| Date | January 2011 |
| Creators | Meng, Xiang |
| Publisher | Glasgow Caledonian University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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